In article <3sl6cr$jca at nuscc.nus.sg>
medp4003 at leonis.nus.sg (Farid John Ghadessy) writes:
>>Hello all,
>>I've been trying to isolate a protein that I'm expressing in the
>periplasm using osmotic shock (Neu and Heppel). The problem is that I
>can't see any bands on an SDS gel after shocking, suggesting I'm doing
>something wrong. I'm only inducing a 10ml culture to test things before I
>scale up but don't seem to be able to liberate ANY proteins from the
>periplasm. My questions.....are certain cell lines less "shockable"
>and/or should I scale up to really see anything.
>>------------------------------------------------------------------------------
Are you sure your expression system is working? I have had variable results
using osmotic shock--I don't know if it is my incompetence though. A more re-
producible method is to make spheroplasts with lysozyme. There is a technique
for this in _CPMB_ under RNA isolation from G- bacteria. However, if your
expression system is working well, you should be able to detect your protein
in a crude lysis on SDS-PAGE. Let me know if you need more info.
--Tom